Vaccination in Leishmaniasis: A Review Article

Leishmaniasis is caused by protozoan Leishmania parasites that are transmitted through female sandfly bites. The disease is predominantly endemic to the tropics and semi-tropics and has been reported in more than 98 countries. Due to the side effects of anti-Leishmania drugs and the emergence of drug-resistant isolates, there is currently no encouraging prospect of introducing an effective therapy for the disease. Hence, it seems that the key to disease control management is the introduction of an effective vaccine, particularly against its cutaneous form. Advances in understanding underlying immune mechanisms are feasibale using a variety of candidate antigens, including attenuated live parasites, crude antigens, pure or recombinant Leishmania proteins, Leishmania genes encoding protective proteins, as well as immune system activators from the saliva of parasite vectors. However, there is still no vaccine against different types of human leishmaniasis. In this study, we review the works conducted or being performed in this field.


INTRODUCTION
eishmaniasis is a vector-borne disease caused by more than 30 species of Leishmania parasites. The disease has a broad clinical picture, ranging from skin lesions to fatal visceral infections [1]. Leishmaniasis is endemic to four continents and more than 98 countries [2] . According to the WHO, 350 million people are at risk for leishmaniasis [2] . Leishmaniasis is found in humans in two main forms: CL and VL. Approximately 58,000 VL cases and 220,000 CL cases are reported annually [2] . The CL is divided into cutaneous, mucocutaneous, and diffused cutaneous types [2] . L. tropica and L. major are the main causes of CL, while L. infantum and L. donovani are the main causes of VL. Different species of rodents in various parts of Iran act as a reservoir for rural CL. These species include Rhombomys opimus and Meriones libycus found in the central and northeast, M. libycus, M. persicus, and M. hurrianae in the south, as well as Tatera indica and Nesokia indica in the west and southwest [3,4] . The Leishmania parasite is transmitted in the Old World, including Europe, Africa, and Asia, by the bite of the female sandfly of the genus Phlebotomus, and in the New World, including America, by Lutzomyia. The L 2 Iran. Biomed. J. 26 (1):  main hosts are vertebrates, and the most commonly infected hosts include humans, dogs, and rodents [5] . The sandfly family consists of five genera and 700 species, of which about 30 species are involved in the transmission of the Leishmania parasite [6] . Table 1 shows the main species of Leishmania that cause human disease. Over the years, many types of research have been conducted on the Leishmania vaccine. In each of these studies, candidate antigens were produced using improved laboratory techniques and various experimental models were examined. An overview of the results from the past to the present investigations can provide a fruitful research strategy for researchers. Meanwhile, such studies have shown that different vaccine administration routes can affect protective immunity. Despite the large number of preclinical vaccine candidates, and approaches designed to emulate this protective response [7] , the successful transition of Leishmania vaccines into human trials has remained elusive, though considerable efforts are underway [8,9] . Therefore, the purpose of this article is to provide a more comprehensive review of the current advances in leishmania vaccine development.

Immunity against leishmaniasis
Macrophages are the primary hosts for Leishmania, but their role in preventing or progressing the disease has been described in T-cell-dependent behavior; however, the fate of the infected macrophages before T cell presence is not well-known [10] . Because specileilized T cells apeare late in the infection, the parasite is able to regulate disease progression in the host.
Parasites can manipulate killing mechanisms of macrophages, at the time of their entry, and stimulate the production of IL-4 and certain disease-stimulating factors by T cells, leading to the progress of the disease and survival of the parasite [11] . As soon as the parasite diverts the CD40 signaling pathway to the preparasitic pathway in macrophages, the interaction between the CD40 ligand presented on activated T cells surfaces and CD40 receptors of infected macrophages cannot activate the anti-parasitic pathway, and probably reaction of T cell-macrophage does not maintain the host [12] . In addition to the host apoptosis, stimulation of parasite apoptosis can be one of the therapeutic goals to increase the effectiveness of antiparasitic drugs. For instance, the study of Sengupta et al. [13] showed that the natural indoloquinoline alkaloid cryptolepine causes a decrease in the cell viability of L. donovani AG83 promastigotes in both time-and concentration-dependent manners by increasing ROS and lipid peroxidation production and decreasing cellular glutathione levels. The results of Roy et al.'s [14] study also indicated that the plant carbazole alkaloid exerts in vitro and in vivo antileishmanial activity by the modulation of redox homeostasis. Furthermore, about inducing host apoptosis, researches have demonstrated the integration of expressional cassettes containing pro-apoptotic genes in Leishmania by transgenic method or downregulating antiapoptotic molecule by miRNA could accelerate the apoptosis process of infected macrophages, restrict the possibility of differentiation and induce more proliferation of Leishmania. These events would result in the expansion of the disease, and the appearance of the lesion [15] . A study by Aghaei et al. [16] signified that the transgenic L. infantum expressing mLLO-BAX-SMAC proteins can accelerate the apoptosis of infected macrophages compared to wild-type Leishmania. It means that transgenic Leishmania is proved to increase the rate of apoptosis in infected macrophages compared to intact strain. Since metacaspases are the key regulators of death or life of parasites, and these proteins do not exist in mammals, they can be considered as targets for fighting against parasitic infections in the future [17] .

Vaccination concepts in leishmaniasis
There are some facts to support the possibility of developing an effective vaccine against CL. However, due to the increased resistance to first-line drugs and the toxicity of second-line drugs, the development of an effective vaccine against the disease is very desirable. The use of vaccines is advantageous over chemotherapy as they induce long-lasting effects and can be administered both in prophylactic and therapeutic modes. Also, the vaccine will not counter the problem of resistance as in the case of chemotherapy [18] . As stated in a study published by Thomaz-Soccol et al. [19] in 2018, the number of patents for leishmaniasis vaccines is 74 in the United States and 36 in Brazil. In Brazil, 20,000 cases of leishmaniasis and more than 3,000 cases of VL, and in India, 8,000 cases of VL are reported annually [20] . Spain and France are still endemic for VL. In France, for example, the prevalence of VL is 0.22 per 100,000 population in the endemic regions [21] . Therefore, vaccination against leishmaniasis is essential in these areas. Moreover, the highest number of patents was reported in that study to be related to the private sector (94 cases), and the lowest was related to cooperation between universities and companies (11 cases); however, universities and noneducational public institutions had 65 and 13 patent cases, respectively [21] . Therefore, the need for more cooperation between public and private institutions seems to be necessary.

Challenges of efficient vaccine design
To date, many attempts have been made to test clinically prepared vaccines in various human trials, but they have been ineffective. It is widely believed that this problem arises from economic and financial pressures [22] . Some studies have shown that using the whole parasite leads to inefficient antigen presentation and anti-Leishmania memory cell development, thus reducing immunity [23][24][25] . Also, preserving central memory T cells does not require the presence of parasites [26] . There may not have been a suitable human adjuvant system for testing these vaccines [27][28][29] . Vaccination provides long-term protection in the absence of attenuated strains such as LdCEN -/-(centrin mutant) or PMMΔ (phosphomannosemutase). This finding was performed in a mouse model and not in humans. Injection of protective antigens in different models or immunotherapy has helped to find the factors involved in increasing anti-Leishmania immunity. One of the major problems facing the vaccine against CL is the fact that despite causing cutaneous disease, the Old and New World parasites, L. major and L. mexicana/L. amazonensis, respectively, are significantly different [30] . There are differences in virulence factors between these species, as well as in the immune responses induced by them. For instance, LPG is a virulence factor for L. major [31] , but not for L. Mexicana [32] . During L. major infection, the protective role of Th1 responses has been established, but L. amazonensis can persist in the presence of Th1 responses and cause minimal disease in the complete absence of T cells [33] . These findings show major, but not well-understood, differences in the immunobiology of parasites that appear to cause the same disease. This matter may have implications for the vaccine development process as the anti-CL vaccine may have different needs for the Old and New World leishmaniases. Therefore, a vaccine against CL caused by L. major might not necessarily be effective against the New World spectrum of diseases, including mucocutaneous and diffuse cutaneous forms. Another challenge for the vaccine is to obtain protection against VL even if it is efficacious against varied forms of CL.

Immunization methods against CL Leishmanization
Adler observed that Lebanese children whose arms have been exposed to infected mosquitoes by their 4 Iran. Biomed. J. 26 (1):  mothers will be protected against severe forms of the disease in the future [34] . This process was not followed because it caused uncontrolled growth of skin lesions and also led to a high prevalence of the disease in people with suppressed immune systems, particularly those with HIV and organ transplants [35,36] . The first method of immunization against leishmaniasis known as "leishmanization" was developed in 1940 and has been used in various countries for several years [37] . This vaccine was discontinued due to its lack of safety and is now limited to the vaccine registered in Uzbekistan and the vaccine used in clinical trials in Iran [38,39] . In this procedure, live and active L. major promastigotes are injected intradermally into the anatomical position of the deltoid muscle. An active ulcer then develops and eventually heals on its own. The result of this method is long-term immunity against rural and urban leishmaniasis. Tables 2 and 3 shows leishmanization experiments in Iran and USSR countries.

First-generation vaccines
These vaccines contain the whole body of the parasite with or without adjuvant [39] . First-generation vaccines replaced leishmanization, and the vaccine is now used in some human trials. These categories include killed, live attenuated, and fractionated vaccines [40] . Table 4 lists the first-generation vaccines with full specifications.

Killed vaccines
This type of vaccine was developed and evaluated by Mayrink et al. in Brazil [41,42] . The result of the leishmanin skin test was satisfactory, but the vaccine had only a 50% protective effect. In Venezuela, Sharples et al. [43] used a mixture of killed L. amazonensis, L. mexicana, and Bacillus Calmet Guerin to treat CL, resulting in a 95% improvement and activation of Th1 immunity [43][44][45] . Studies in Brazil have shown that a mixture of killed L. amazonensis with half a dose of meglumine antimoniate is very effective in treating CL [46] . According to a study conducted in Ecuador, a proportion of L. brasiliensis, L. guianensis, and L. amazonensis provided favorable protection against CL [47][48][49] . Two studies in Iran have shown that autoclaved L. major vaccine with BCG is safe but does not provide promising immunity against CL [50,51] . The results of a study by Mahmoodi et al. [52] revealed that cases who received the ALM + BCG vaccine had a higher stimulation index and IFN-γ levels than those who received BCG alone or in the control group. The results of this study showed that the induction of Th1 immune response in volunteers who received the vaccine was much lower than those with or without a previous history of leishmaniasis, and it was assumed that these individuals became immune [52] . Th1 is activated in L. major infection, but L. amazonsensis can remain active in the presence of Th1 and can reduce the T cell response. Therefore, the vaccine made for L. major is neither effective for another leishmaniasis nor VL. In general, vaccination with killed Leishmania promastigotes could be considered as a safe and economical treatment; nevertheless, further trials aiming at the evaluation of different adjuvants potentially pave the way for more efficient vaccines [53] .

Live attenuated vaccine
These vaccines are currently the gold standard. In attenuated live vaccines, the parasite is both nonpathogenic and superior to killed vaccines [54] . Methods of preparing attenuated live parasites include long-term in vitro culture [55] , use of temperature sensitivity [56] , gamma radiation [57] , chemical mutagenesis [58] , and culture with gentamicin [59] . Titus and co-workers [60] developed a live attenuated vaccine by knocking down certain Leishmania genes. Examples in this regard are the DHFR-TS [60] and the lp2 gene, which encodes an enzyme, transports guanosine diphosphate mannose to the Golgi apparatus [61][62][63] , the lpg2 mutant from L. mexicana [64] , the CP (cpa and cpb) from L. mexicana [65,66] , the SIR2 from L. infantum [67] , and the BT1 gene from L. donovani [68] .

Suicidal cassettes
Muyombwe et al. [69] followed a method of producing a vaccine against leishmaniasis, which was to induce suicide genes. This method is performed by inducing drug-sensitive genes. They used a combination of thymidine kinase and gancyclovir against L. major and finally using gancyclovir treatment, partial to complete protection was achieved [70,71] . Besides, the susceptible strain of L. major, which contained the altered thymidine kinase HSV-1 (tk) gene and the cytosine deaminase gene from Saccharomyces cerevisiae (cd), increased susceptibility to gancyclovir and 5-fluorocytosine. L. major infection recovered within two weeks of treatment with either drug alone or in combination with ganciclovir and 5fluorocytosine [70,71] .

Fractionated vaccine
This kind of vaccine is advantageous due to its high purity and yield. Several molecules, either membrane proteins, such as HASPB1 and A2 protein, or soluble fractions of the parasite, i.e. PDI, TPI, elF-2, aldolase, enolase, P45, tryhpanothione reductase, and   [72] .

Second-generation vaccines
Second-generation vaccines are based on synthetic or recombinant subunits and genetically modified Leishmania strains, recombinant bacteria, or viruses carrying Leishmania antigen genes [73][74][75] . A summary of these vaccines against Leishmania is given in Table 5.

Vaccines based on nonpathogenic Leishmania
In 2015, Katebi et al. [76] showed that vaccination with L. tarentolae-PpSP15 in combination with CpG as a prime-boost modality confers strong protection against L. major infection, which was superior to other vaccination methods discussed in the present study. This approach represents a novel and promising strategy for vaccination against Old World CL. In 2014, Zahedifard et al. [77] demonstrated the effect of a novel combination of protective parasitic antigens created by L. tarentolae, together with sandfly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective effect of different DNA/Live and Live/Live prime-boost vaccination with live L. tarentolae expressing CPs (type I and II, CPA/CPB) and PpSP15 from Phlebotomus papatasi, were tested in BALB/c and C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when the mice primed with PpSP15 DNA and then received PpSP15 DNA and live recombinant L. tarentolae as a booster [77] . In 2015, Shahbazi et al. [78] vaccinated outbreed dogs with a prime-boost regimen based on recombinant L. tarentolae expressing the L. donovani A2 antigen, along with CP genes (CPA and CPB -CTE ) and evaluated its immunogenicity and protective immunity against L. infantum infectious challenges. 6 Iran. Biomed. J. 26 (1): 1-35 They showed that vaccinated animals produced significantly higher levels of IgG2, but not IgG1, as well as IFN-γ and TNF-α, but low IL-10 levels, before and after challenge as compared to control animals. Protection in dogs was also associated with a strong DTH response and a low parasite burden in the vaccinated group. Overall, immunization with recombinant L. tarentolae A2-CPA-CPB -CTE proved to be immunogenic and induced partial protection in dogs, hence representing a promising live vaccine candidate against canine VL [78] . In 2013, Saljoughian et al. [79] used a tri-gene fusion recombinant L. tarentolae expressing the L. donovani A2 antigen, along with CPs, as a live vaccine. Their results showed that immunization with both prime-boost A2-CPA-CPB -CTE -recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with the Th1 immune response due to the high levels of IFN-γ production before the challenge, leading to a significant increase in the IFNγ/IL-10 ratio compared to the control groups. In addition, this immunization induced an elevated level of IgG1 and IgG2a humoral immune responses. Protection in mice was also associated with a high NO production and low parasite burden. Altogether, these results indicate the potential of the A2-CPA-CPB -CTErecombinant L. tarentolae as a safe live vaccine candidate against VL [79] .

Lactococcus lactis as a tool for Leishmania vaccination
L. lactis is a well-defined, food-grade lactic acid bacterium commonly known as generally recognized as safe status. A better understanding of this bacterium at a molecular level has led to the development of unprecedented genetic tools that enable the expression of heterologous proteins. Consequently, the ability of L. lactis to express and deliver these proteins to eukaryotic hosts offers a promising approach to achieve potent treatments for various diseases. Currently, 13 genera have been classified under the lactic acid bacterium group, including Lactococcus, Lactobacillus, Streptococcus, Pediococcus, Paralactobacillus, Enterococcus, Carnobacterium, Lactosphaera, Leuconostoc, Oenococcus, Tetragenococcus, Weisella, and Vagococcus [80] . In 2012, Hugentobler et al. [81] described the generation of L. lactis(alr-) strain as the vector expression of the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall or co-expressing mouse IL-12. They showed that oral immunization using live L. lactis, secreting both LACK and IL-12, was the only regimen that partially protected BALB/c mice against the next L. major challenge. This issue highlights the importance of temporal and physical proximity of the delivered antigen and adjuvant for optimal immune priming by oral immunization. In 2019, Torkashvand et al. [82] expressed F1S1 fusion protein, including the N-terminal region of S1 subunit of PT and FHA type1 immunodominant domain by L. lactis, and evaluated its immunogenicity. Based on their results, mice immunized with LL-F1S1 produced significant levels of specific IFN-γ compared to controls and DTaPimmunized mice. The F1S1-specific IgG antibody response was lower in LLF1S1-immunized mice, while the IgG2a/IgG1 ratio was higher in this group compared to the DTaP-immunized mice. In 2020, Davarpanah and co-workers [83] explained that PpSP15 is an immunogenic salivary protein from P. papatasi. Immunization with Lactococcus lactis expressing sand fly PpSP15 salivary protein has been shown to protect against L. major infection. In their study, BALB/c mice were challenged with L. major plus P. papatasi salivary gland homogenate. Evaluation of footpad thickness and parasite burden displayed a delay in disease development and reduced the number of parasites in PpSP15 vaccinated animals as compared to the control group. In addition, vaccinated mice exhibited Th1 type immune responses. Importantly, immunization with L. lactis-PpSP15-EGFP cwa enhanced long-term memory in mice, which lasted for at least six months.

Third-generation vaccines DNA vaccines
These vaccines contain plasmid DNA, which, after injection, encodes foreign proteins, leading to the synthesis of endogenous proteins and the production of specific immune responses [84] . DNA vaccines promote both cellular and humoral immunity [85,86] . DNA vaccines can come in many forms, including recombinant proteins [ . DNA vaccines are made up of heterologous DNA (usually a plasmid) that produces antigenic proteins. These DNAs are supplied by vectors that allow them to be expressed in eukaryotic cells [84] . Advantages of DNA vaccines include (1) fast, simple, and cheap large-scale production, (2) no need for low temperature, transportation, and storage, and (3) the ability to provide long-term protection against multiple strains of Leishmania. The main concern with these vaccines is the risk of parasite DNA entering the mammalian genome. This problem carries the potential risk of cancer and autoimmune diseases [84] . A summary of DNA vaccines is given in Table 6 and the best recombinant salivary candidates is shown in Table 7.

Vaccine products for potential licensing
There are no licensed products yet, but potential candidates could be as follows [108] : (1) a mixture of recombinant proteins (Leish F1, Leish F2, and Leish F3), designed by Infectious Disease Research Institute (Seattle, USA), is currently in the second phase of a clinical trial; (2) recombinant proteins from Leishmania and sandfly saliva (phlebotomus) antigens, designed by Sabin product development partnership (Washington, USA) [19] , is now in the preclinical phase. FML-QuilA (Leishmune®), a protein vaccine, was the first approved vaccine in Brazil in 2003. However, the license to produce and sell the vaccine was suspended in 2014, and its production was stopped by factories. The reason for discontinuation was the incompleteness of the third phase of the trial. There are presently two vaccines against canine VL: A2 Leishmanial Ag from Brazil and Li ESP/QA-21 from France [19] .

DISCUSSION
Vaccines are undoubtedly the most effective way to control diseases. For this reason, the development of safe and cost-effective vaccines, particularly for the diseases with no available vaccine (e.g. leishmaniasis) is an important global public health priority. A major barrier to the development of an effective vaccine is related to the discrepancies between the animal models and human diseases, as well as the transition of the research from the laboratory to the field. Additionally, many questions related to the immune responses and maintenance of immunological memory during an active Leishmania infection have not yet been extensively studied or answered. This article tried to focuse on the latest information related to antileishmanial vaccine development and also major problems with vaccine development and implementation. Candidates for the Leishmania vaccines include leishmanization, as well as the first-, second-, and third-generation vaccines. The development of an effective Leishmania vaccine poses many challenges, mainly related to the complexity of the immune responses to Leishmania, insufficient knowledge of Leishmania pathogenesis, and the discrepancy between the Old and New World parasites. It appears that a successful vaccine will most likely be composed of several antigens rather than a single one, which suggests that combination vaccines and welldeveloped adjuvants, such as Leish-111f and MPL-SE, have the best chances of success. Further clinical trials provide more information on the success of these combination vaccines. In addition, the poor efficacy of the killed and subunit vaccines makes the use of live-attenuated vaccines the next best alternative [109] .
Many questions about antileishmanial immunity in humans have not yet been answered. It is not clear whether parasite persistence is required to maintain immunity in humans. Although parasite persistence in humans is unknown, it is worth noting that an experimental mouse model has revealed the persistence of the parasite following infection [110] . A study has been shown that the absence of parasites leads to the loss of immunity, implying that continuous antigen presence is needed for complete protection [22] .
In contrast, another study in a mouse model has revealed that the maintenance of memory T-cells is independent of parasite persistence, and therefore vaccination with non-persistent strains and nonpersistent, attenuated strains such as LdCEN -/or ∆PMM results in long-term protection [22] . In general, due to the complex nature of the immune response to Leishmania, it is crucial to better understand the determinants of T-cell for long-term immunity and the immunity factors affecting antileishmanial immunity before the development of an effective vaccine. Our understanding of the determinants of T cells is required for long-term protective immunity, although there are still many unknowns. It is hoped that new strategies will be developed to produce effective T-cell vaccines.   The most important thing to consider before making a Leishmania vaccine is to determine the best immunity correlations, as well as to develop efficient delivery systems and improved adjuvants. According to advanced research in parasite immunology and genetic engineering, an effective anti-Leishmania vaccine not far away. In this study, data extraction was performed by two researchers, which may result in errors. Searching for English language and scientific articles in other languages, which may have valuable information from Africa, the Middle East, and Asia, were limited. Despite these limitations, the present study attempted to review the content of credible articles that lead to clear and up-to-date information on the performance and effectiveness of various vaccines designed against leishmaniasis.
Given the global importance of leishmaniasis, decisive measures must be taken to prevent this disease with social impacts. It seems that one of the effective ways to control leishmaniasis is immunization of people living in endemic areas of the disease. In this review, it was found that an effective vaccine against leishmaniasis is not yet available, and scientists in this field have chosen different methods to produce such a vaccine. The results of these efforts have been the production of three different generations of Leishmania vaccines. In any case, summarizing the results of these studies and trying to clarify as much as possible the ambiguities in the immunity of leishmaniasis and especially the interaction of the parasite with host cells will help to advance in the right direction. Understanding more about the unknown mechanisms of the behavior of the parasites inside the host body will persuade us to produce an effective vaccine against the disease.